PROJECT SUMMARY The long-term goal of this research is to understand how Mycobacterium tuberculosis toxin-antitoxin systems are regulated to contribute to tuberculosis pathogenesis. The specific goal of this R21 research proposal is to investigate our hypothesis that Ser/Thr phosphorylation of VapB antitoxins by receptor-type Ser/Thr protein kinases represents a novel mechanism by which VapC toxins can be regulated in response to environmental signals. VapC toxins are the majority of all TA toxins in M. tuberculosis and have been implicated in proteome remodeling, growth arrest, antibiotic tolerance and ability to survive a range of stresses that are relevant for tuberculosis pathogenesis. The established role of VapB antitoxins in sequestering cognate VapC toxin proteins and autoregulating vapBC gene expression, together with our recent finding of significantly decreased phosphorylation of several VapB proteins in the setting of specific kinase depletion, provide the premises for this research. This research proposal has two Aims. In Aim 1 we will determine the effects of phosphorylation of two VapB antitoxins on i) the growth of M. tuberculosis expressing these antitoxins together with their cognate VapC toxins, and ii) the molecular interactions of these VapB toxins with their cognate VapC proteins and their cognate promoters. In Aim 2 we will investigate the effects of phosphorylation of these VapB antitoxins on VapC enzyme activity. This research will test our hypothesis that VapB Ser/Thr phosphorylation may be a means to regulate VapC activity and will begin to characterize the molecular mechanism(s) by which this regulation occurs. Results from this research will provide the foundation for further investigation into how Ser/Thr phosphorylation of VapB antitoxins can function to transduce signals to control the activity of VapC toxins that are relevant for M. tuberculosis growth control, stress tolerance and tuberculosis pathogenesis.